The organization of the eukaryotic genome into chromatin allows the cell to regulate all DNA-dependent processes. Current models of chromatin structure hold that it exists in four levels, similar to protein structure. The secondary structure of chromatin is the folding of chromatin into structures such as the 30 nanometer fiber, which is mediated by local interactions between nucleosomes on the same DNA strand. Secondary structure is considered to be one of the strongest mechanisms of transcriptional repression, during which interactions between neighboring nucleosomes block events such as transcription factor binding and polymerase elongation. However, studying chromatin structure at this level has been the most difficult. Recent studies of chromatin purified from cells have failed to observe regularly folded chromatin fibers, casting doubt on the existence of 30 nanometer fibers. Genomics and microscopy methods, which characterize chromatin in its cellular context, have been unable to reach resolutions necessary for examining secondary structure, leading chromatin structure below the kilobase pair level to be frequently referred to as ?a blind spot.? The recent development of a genomics technique called Micro-C has broken this technical barrier in Saccharomyces cerevisiae. Micro-C modifies the well-established Hi-C protocol by using Micrococcal nuclease to digest crosslinked chromatin down to nucleosomes, ligating DNA between crosslinked nucleosomes, and then identifying ligated sequences. Whereas Hi-C methods reach resolutions of 1-4 kilobases at best, Micro-C provides maps of inter-nucleosomal interactions at 150 base pair single-nucleosome resolution. Micro-C experiments in exponentially growing cultures discovered secondary structure in the form of disordered ?crumpling? interactions between nucleosomes in the same gene, but found little evidence for a folded chromatin fiber. However, chromatin folding is not predicted to be a prevalent feature of actively growing yeast. A life stage during which secondary structure is expected to play a more significant role is quiescence (Q), a reversible phase in which cells enter a long-lived, non-replicative, and transcriptionally inactive program. Previously published and preliminary data suggest that a global increase in chromatin folding controls transcriptional repression during Q, and implicate the Isw2 chromatin remodeling enzyme in mediating this repressive structure. In the work described in this proposal, I will test these hypotheses by using Micro-C to map chromatin structure in log and Q cells genome-wide. Once Q cells are established as a model of functional secondary structure, I will be able to uncover the mechanisms of chromatin folding and determine its role in transcriptional repression. I will also investigate how Isw2 affects secondary chromatin structure, and test the model that an increase in chromatin folding during Q directs Isw2 targeting. These experiments will fill a critical gap in our knowledge of chromatin structure, be the first to determine the mechanisms and functions of chromatin folding within cells, and establish relationships between chromatin structure and remodeling.